Diacetyl is a high value flavor compound, which contributes to the buttery aroma of many fermented foods, including cheese, butter and butter milk. Lactococcus lactis has a native metabolic pathway leading to the synthesis of meso-2,3-butanediol. This pathway, starting from glycolysis employs two molecules of pyruvate which are condensed to α-acetolactate by the enzyme α-acetolactate synthase (ALS). Acetolactate, which is unstable, is converted to diacetyl by non-enzymatic oxidative decarboxylation. However, diacetyl accumulation is extremely low since most of the acetolactate flux is converted to acetoin by the activity of an α-acetolactate decarboxylase (ALDB), which in turn can be converted to meso-2,3-butanediol. The major fermentation products produced by Lactococcus lactis include lactate, ethanol and acetate.
In contrast to bacterial strains derived from pathogens, some lactic acid bacteria, in particular Lactococcus or Lactobacillus species are particularly suitable for production of food grade diacetyl, since they have “generally recognized as safe” (GRAS) status. In addition, they have a high glycolytic flux, and well-characterized metabolic pathways. Under normal conditions, Lactococcus or Lactobacillus species are fermentative and more than 90% of glucose is converted to lactate. Although diacetyl levels are very low in wild type lactic acid bacteria, the application of metabolic engineering strategies makes these bacteria suitable candidates for developing genetically modified strains capable of homo-diacetyl production. There exists a need for lactic acid bacteria having GRAS status that can produce diacetyl from glucose as substrate and where the conversion of glucose to diacetyl has a high mol/mol yield.